Effects of the combination of melatonin and N-acetylcysteine on the inflammatory response in a rat model of cerebral ischemia

Abstract

Stroke is the second leading cause of death and long-term damage globally. Inflammation is a significant factor in the onset of ischemic stroke. This study investigated the simultaneous administration of melatonin and N-acetylcysteine (NAC) on inflammation in rat cerebral ischemia. First, 30 male Wistar rats were randomly divided into five groups (n = 6), including the sham group without ischemia, the ischemic group, and the ischemic groups treated with NAC, melatonin, and NAC + melatonin, respectively. To induce ischemia, a silicone-coated monofilament was placed from the common carotid artery towards the middle cerebral artery and stained for 60 min. The rats were treated by administering NAC (50 mg/kg), melatonin (5 mg/kg) and the combination of NAC + melatonin by intraperitoneal injection after ischemia induction. The animals were assessed for sensory-motor activity at 24 and 72 h. Following sacrifice, the rats' brain was dissected to estimate infarct volume after triphenyltetrazolium chloride (TTC) staining. Inflammatory parameters were then analyzed through gene expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and nucleotide oligomerization domain (NOD)-like receptor family with pyrin domain 1 and 3 (NLRP1 and NLRP3). The results showed a significant decrease in mRNA expression of the target genes in the rats treated with NAC + melatonin compared to the ischemic group (p < 0.05). The group that received the combined treatment exhibited enhanced sensory-motor function and a reduced brain infarct volume compared to the other groups (p < 0.05). In summary, the combined use of NAC and melatonin has shown promise in enhancing neurobehavioral function and decreasing the volume of cerebral infarction by regulating the inflammatory signaling pathway.

Keywords: Cerebral ischemia; Cytokines; Inflammasomes; Inflammation; Melatonin; N-acetylcysteine (NAC).

Graphical abstract

Fig. 1

Triphenyltetrazolium chloride (TTC) staining was conducted on five consecutive coronal sections of the brain 72 h post-ischemia induction in the left hemisphere to evaluate the infarct volume. In both the ischemia and treatment groups, arrows pointed to the white or penumbral regions, indicating areas affected by ischemia, while the sham control group displayed a red cortex. Notably, the melatonin + NAC group demonstrated a significant decrease in infarct volume when compared to the ischemic group and other treatment cohorts (n = 3).

Fig. 2

Hematoxylin and eosin (H&E) staining was performed 72 h following ischemia induction in the left hemisphere of the ischemic group. Observations made under a stereo microscope (a) indicated tissue damage in the left hemisphere, marked by a red line. Further examination with a light microscope (b & c) confirmed tissue injury in the left hemisphere (c) while the right hemisphere maintained an intact tissue structure (b).

Fig. 3

Reverse-transcription quantitative polymerase chain reaction analysis of mRNA expression of nuclear factor kappa B (NF-κB) (A), tumor necrosis factor alpha (TNF-α) (B), interleukin-lβ (IL-1β) (C), nucleotide oligomerization domain (NOD)–like receptor family pyrin domain-containing 1 (NLRP1) (D), and NLRP3 (E) in the brain tissue of sham, ischemic, ischemic treated with NAC (Ischemic + N), ischemic treated with melatonin (Ischemic + M) and ischemic treated with NAC + melatonin (Ischemic + N + M) groups 72 h post-ischemia induction. Each value represents the mean ± SD (n = 6 per group). Statistical analysis revealed significant differences between groups: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.